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IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS)
e-ISSN: 2278-3008, p-ISSN:2319-7676. Volume 6, Issue 6 (Jul. – Aug. 2013), PP 19-23
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Dietary Supplementation with Calcium in Healthy Rats
Administered with Artemisinin –based Combination Therapy and
Risk of Cardiovascular Disease
Emmanuel O Ajani1x, Fisayo A Bamisaye1, Ogunsola Margaret O2, Bamidele A
Salau3
1
Department of Biosciences and Biotechnology, College of Pure and Applied Sciences, Kwara State University,
Malete, P. M.B. 1530 Ilorin, Kwara State, Nigeria
2
Department of Biochemistry, Faculty of Science, Bowen University, Iwo, Osun State Nigeria.
3
Department of Chemical Sciences, College of Natural Sciences, Redeemers University, Km 46, Lagos/Ibadan
Expressway, P. M. B. 3005, Redemption city, Mowe, Ogun State, Nigeria.
Abstract: Reports on the role of calcium on predisposition to cardiovascular disease have been rather
inconsistent while studies on its interaction with other medications are ongoing. We therefore investigated the
effect of separate and combine administration of calcium supplement with artemisinin-based combination drug
on hepatic and serum lipid profile. Thirty two male wistar rats were randomly assigned into four groups of
eight rats each. The control (group A) received normal saline. Group B and D were placed on 10mg/Kg
calcium twice daily for four weeks. On the thirtieth day, therapeutic dose of artemisinin-based combination was
simultaneously administered to group C and group D twice daily for three days. All the rats were then
sacrificed after 12 hours fasting, blood was withdrawn and the liver removed and homogenized in an
appropriate buffer. Biochemical analysis showed no significant (p> 0.05) variation in hepatic triaacylglycerol
in all the treated groups whereas calcium supplementation was observed to induce a significant (p< 0.05)
reduction in hepatic cholesterol. Significant elevations due to calcium supplementation were also observed in
serum total cholesterol, LDL cholesterol level and atherogenic risk index with a concomitant reduction in serum
HDL cholesterol. No significant change was observed in serum total cholesterol, triacylglycerol and serum
lipoproteins in all other treatment groups. Our study suggests that calcium supplementation may predispose to
cardiovascular disease and that its co administration with ACT may not aggravate nor reduced the
predisposition risk.
Key words: Lipid profile, cardiovascular disease, calcium, ACT, malaria, Rat.
Submitted date 04 June 2013
Accepted Date: 10 June 2013
I.
Introduction
Calcium is the fifth most abundant element in the human body, with ∼1000 g present in adults. It plays
a key role in skeletal mineralization, as well as a wide range of biologic functions such as nerve depolarization,
signal transduction, muscle contraction, maintenance of electrochemical gradient across the plasma membrane
and neuronal function [1]. It is an essential element that is only available to the body through dietary sources.
Current dietary calcium recommendations range from 1000 to 1500 mg/d, depending on age [2]. It has been
noted [3] that some individuals, particularly the elderly may require calcium supplements in order to achieve the
recommended dietary calcium intake. An individual requirement for calcium is dependent on the state of
calcium metabolism. This in turn is regulated by three main mechanisms: intestinal absorption, renal
reabsorption, and bone turnover. These in turn are regulated by a set of interacting hormones, including
parathyroid hormone (PTH), 1,25-dihydroxyvitamin D [1,25(OH)2D], ionized calcium itself, and their
corresponding receptors in the gut, kidney, and bone.
Malaria is one of the worst health problems in this decade. Globally, the disease takes the heavy toll of
more than one million lives every year. The worsening problems of drug resistance in many parts of the world
and the limited number of antimalarial drugs available have led to increasing difficulties for adequate disease
management. Artemisinin and its derivatives are among the most effective antimalarial drugs known today.
They rapidly cure even drug-resistant falciparum infections. Artemisinin-based combination therapies (ACTs)
are being used for treatment, instead of mono-therapies, to delay the emergence of resistant strains
of Plasmodium falciparum to this vital class of drugs. ACTs are increasingly being adopted as first-line
treatments in malaria-endemic regions of the world that are afflicted with P. falciparum strains resistant to
conventional antimalarial drugs.
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Dietary Supplementation With Calcium In Healthy Rats Administered With Artemisinin –based Combination
Many authors have reported on the metabolic effects of artemisinin and on the pharmacological effects
of its interaction with some other medications [4, 5]. It has been suggested that high doses of artemisinin drugs
can produce neurotoxicity, prolongation of the QT interval, bone marrow depression, with the possibility of long
term toxicity in human beings [6]. However, no report on the pharmacological effects of its interaction with
calcium has been cited. A study reported that calcium and calcium containing products interact with drug such
as rosephine and cause dangerous deposits in the heart, lungs and kidneys [7]. Another study reported that
calcium can decrease the absorption of other drugs [8] with serious consequence.
Alterations in dietary conditions may result in a wide variety of physiological and pathological changes
in the body system. This may include alterations in the internal milieu and the extracellular fluid which
surround individual cells [9]. Consequently, this may result in changes in concentration, composition, and
configuration of membrane transporters. The potential effects of inadequate or excessive calcium supply on
cardiovascular disease (CVD) are receiving growing attention A number of prospective epidemiologic studies
have examined the relationship between dietary calcium intake and CVD incidence or mortality in middle-aged
and older adults, however, the results were inconsistent [10-12]. A report by Lartey-Rowser [13] noted that the
relationships between calcium intake and body mass index, total cholesterol, high-density lipoprotein and lowdensity lipoprotein in African-American adults vary between genders (from being a positive relationship to a
negative relationship depending on the factor). What researchers do not know is whether this process is
influenced by the amount of calcium a person takes in each day from supplements.
In the present study we have investigated the modulating effect of calcium supplementation and the effect of coadministration of calcium with artemisinin- based combination drug on lipid profile of both the plasma and the
liver. This was done as an attempt to understand the role of separate and combined administration of these
medications in predisposition to cardiovascular disease.
II.
Materials and methods
2.1
Drug
Leonart® (Bliss GVS Pharma Ltd. India) composed of 80 mg Artemether and 480mg lumefantrine and
calcium compound (GNLD Int. Spartan, South Africa and Lagos, Nigeria) composed of 300mg calcium
glycinate) was obtained from Boorepo Chemist, Osogbo, Nigeria.
2.2
Experimental subjects
Thirty two (32) male wistar rats weighing 100-150g were obtained from the animal facility centre,
University of Ibadan, Nigeria. All animals were kept in individual cages in an environmentally controlled room
with a 12 h light/12 h dark cycle. The animals were placed on standard rat feed (Ladokun feed, Ibadan, Nigeria)
and clean drinking water ad libitum. They were all acclimatised for one (1) week. The care of the animals was
in accordance with the U.S. Public Health Service Guidelines [14] and approved by the Ethics Committee,
Kwara State University, Malete, Nigeria.
2.2.1
Grouping of animals
The rats were randomized into four (4) groups (each containing 8 rats), labeled and treated as indicated
below.
Group A: Normal control
Group B: Calcium supplement
Group C: Artemisinin- based combination drug
Group D: Calcium supplement + Artemisinin- based combination drug
2.2.2
Experimental procedure and administration of Drugs:
The calcium supplement was administered (to rat in group B and D) at a therapeutic dose of 10mg/kg
body weight twice daily while artemisinin- based combination was administered at a therapeutic dose of
2.67mg artemether/16mg lumefantrine per Kg body weight twice daily (to rats in group C and D). All drugs
were administered in an aqueous suspension by oral galvage. The drug suspensions were continuously agitated
in order to deliver the drug homogenously to the animals. The calcium was first administered to the rats for four
(4) weeks. On the thirtieth day, artemisinin- based combination drug was simultaneously administered (group C
and D) for another three (3) days. After the last administration, the rats were fasted for twelve (12) hours and
were then sacrificed. Blood was drawn from their eye vein and delivered into lithium heparinised tube by
inserting a capillary tube. Plasma was then prepared by centrifugation at 3000 rpm for 10 minutes. The liver
was also removed, clean of blood, weighed and homogenised.
2.3
Analytical procedure
Plasma triacylglycerol, total cholesterol, and HDL-c were determined by colorimetric methods after
enzymatic reaction with peroxidase (Microlab 300, Leatherland, Dieren, UK). Low-density lipoprotein
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Dietary Supplementation With Calcium In Healthy Rats Administered With Artemisinin –based Combination
cholesterol was estimated by the Friedewald et al [15] formula, which is reliable when triacylglycerol levels are
lower than 400 mg/dL. Hepatic triacylglycerol, and total cholesterol were measured using established methods
[16-18]. Hepatic lipid was extracted using the method of Folch et al [16] and was gravimetrically determined.
Liver triacylglycerol was measured using the method described by Biggs et al. [17] and total cholesterol, using
the method described by Zlatkis et al.[18]. Very low Density Lipoprotein (VLDL) was estimated by
mathematical computation using friendship relationship VLDL = TG ÷ 2.2. Atherogenic index (A.I.) was
calculated using the formula of Abot et al.[19] and coronary risk index (C.R.I.) was obtained by the method of
Alladi et al. [20].
2.4
Statistical analysis
Data analyses were performed using SPSS software (SPSS 10.0 for Windows, SPSS Inc, Chicago, IL).
All data are expressed as mean ± SEM. Analysis of variance was used to test for differences between the
groups. Duncan’s multiple range test was used to determine the significance of differences among the mean
values at the level of p< 0.05.
III.
Result
Table 1 presents the result of hepatic lipids determined after treatment. Separate administration of
calcium supplement was observed to decrease liver total cholesterol from the normal control value of 4.58±0.10
to 2.61±0.12mmol/L but does not alter the triacylglycerol level significantly (p> 0.05). Neither separate
administration of ACT nor its combined administration with calcium significantly alters the liver total
cholesterol and triacylglycerol level.
Shown in Table 2 is the result of the effect of treatment on plasma lipids. Calcium supplementation
was observed to have significantly increased the plasma total cholesterol from the normal control value of
6.02±0.10mmol/L to 7.94±0.22mmol/L. Separate administration of ACT and combined administration of ACT
with calcium however did not significantly affect plasma total cholesterol. The observed plasma cholesterol of
6.28± 0.18mmol/L following separate administration of ACT and the observed value of 7.47± 0.20mmol/L
when co administered with calcium were not different from each other. A similar trend in triacylglycerol level
was observed with calcium administration, however the observed triglyceride level following separate
administration of ACT and when co administered with calcium were not different from that of the normal
control value.
The observed trend in HDL-c as shown in Table 2 indicates that whereas separate administration of
calcium significantly lowered plasma HDL-c from the normal control value of 3.30±0.39mmol/L to
1.94±0.31mmol/L, separate administration of ACT and its combined administration with calcium does not
significantly altered plasma HDL-c value. A raised plasma LDL-c was observed with calcium treatment, ACT
administration however was not observed to affect plasma LDL-c value. The observed value of 2.49±
0.07mmol/L was not different from the observed value of 2.13±0.21mmol/L when ACT and calcium were co
administered. Neither separate administration of calcium and ACT nor their combined administration
significantly affects plasma VLDL-c value.
Table 3 indicates that calcium supplementation increased artherogenic risk index but does not
significantly altered the coronary risk index. Separate administration of ACT and its combined administration
with calcium however do not caused any variation in both artherogenic and coronary risk indexes.
IV.
Discussion
High levels of total cholesterol and LDL-c are major risk factors for coronary diseases, whereas
increased HDL-c is associated with a decrease in coronary disease risk [21]. Another previous study also
reported that elevated triacylglycerols could increase the incidence of coronary heart disease [22]. Data
generated from our study showed that calcium supplementation increased levels of serum total cholesterol,
triacylglyserol and LDL-c. These results suggest that calcium supplementation may predispose to cardiovascular
disease risk. The result is further supported by the increased artherogenic risk observed in the study when the
subjects were placed on calcium supplements. It is well known that HDL-c plays an important role in the
transport of cholesterol and cholesterol ester from the periphery (away from the tissues) to the liver by the
reverse cholesterol transport pathway where they are further converted to bile acids. Therefore, the observed
decrease in serum HDL-c in the rats administered with calcium may be related to the increase serum cholesterol
and subsequently reduced hepatic cholesterol synthesis through depression of HMG-CoA reductase [23]. This
result corroborates previous laboratory studies [24, 25]. Calcium may affect the risk of developing CVD through
multiple mechanisms including blood cholesterol, insulin secretion and sensitivity, vasodilation, inflammatory
profile, thrombosis, obesity, and vascular calcification [25]. In a previous study by Wang et al. [26], it was
reported that calcium may builds up in plaque, the cholesterol-filled pockets that grow inside arteries like tiny
pimples. The consequent effect of narrowing arteries as a result of cholesterol deposition is that the plaque can
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Dietary Supplementation With Calcium In Healthy Rats Administered With Artemisinin –based Combination
choke off the supply of blood to heart muscle and other vital tissues. If a plaque bursts open, it can trigger a
heart attack, stroke, or sudden cardiac arrest. Based on data from our study therefore, we thus opined that over
time, calcium can accumulate in arteries. This will makes them stiffer and less responsive to the demands of the
body and thus contributing to high blood pressure, angina (chest pain with exertion or stress), and heart failure.
The LDL or harmful cholesterol transports free cholesterol and cholesterylester into the tissues where
they accumulate and foster arteriosclerosis. The observed low level of hepatic cholesterol in rats placed on
calcium supplement reported in the present study correlated with the decrease HDL cholesterol and increased
LDL cholesterol observed in the same group of rats. Patients with arterial disease can have elevated VLDL or
LDL or both [27]. Although our study with calcium supplements noted an increased LDL-cholesterol, the
VLDL cholesterol was normal. We also noted that though separate administration of ACT does not contribute
to alteration in these parameters, its co administration with calcium does not also prevent elevation of both total
cholesterol of the plasma and plasma LDL cholesterol values elicited by calcium supplement. Data from this
study therefore suggests that Treatment of an individual with artemisinin- based combination drug when placed
on calcium supplement may also predispose to cardiovascular diseases. No alteration was observed in the
hepatic lipid with separate and combined administration of ACT with calcium. Our findings in this study
support the previous report of Georgwill et al.[28] which noted in a toxicological study that artemisinin
combinations causes no significant treatment related histopathological changes in the heart, liver and kidney of
all treated rat groups as the tissues showed normal cytoarchitecture comparable to that of the control.
Body cells extract cholesterol from the blood by means of receptors on their surfaces. These receptors
bind with the LDL particles (and their attached cholesterol) and draw them from the blood into the cell. When
more LDL particles are captured by the receptors of the body cells, the formation of more receptors on that
cell’s surface is inhibited, thus lowering its future intake of cholesterol. Fewer receptors on the body cells mean
that less cholesterol is ingested by the cells and that more remains in the blood stream. This thus increases the
risk of cholesterol (LDL) accumulating in the internal walls of the blood vessels [29]. The increase in LDL
observed in our study with calcium supplementation we opined, may increase the rate of transportation of free
cholesterol and cholesterylester into the tissue where they could accumulate and foster arteriosclerosis. This we
suggest may be the mechanism by which increased calcium supplementation predispose to cardiovascular
disease. Also the decrease in HDL may have caused a reduction in the rate of transportation of free cholesterol
and cholesterylester from tissues to the liver where it is converted to bile acid [28, 30].
V.
Conclusion
The result of this study further validate some of the initial studies which reported that increased
calcium supplementation can predispose to cardiovascular diseases and disagree with studies that reported that
calcium supplementation does not correlate with cardiovascular disease. Again, we are reporting based on the
result of the present study that administration of therapeutic dose of artemisinin-based drug and its co
administration with calcium may not increased the tendency of calcium supplement predisposition to
cardiovascular disease.
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Table 1: Effect of treatment on hepatic cholesterol and triglyceride
Group/ Treatment
A (Normal control)
B (Calcium)
C (Artemisinin combination)
D (Artemisinin + Calcium)
Note:



A
(Normal
control)
B (Calcium)
C (ACT)
D
(Calcium
+ACT)
Total
cholesterol
6.02± 0.10a
7.94± 0.22b
6.28
± 0.18a
7.47± 0.20b
Plasma lipid (mmol/L)
Triglyceride HDL-c
LDL-c
VLDL-c
1.37±0.26a
3.30±0.39a
2.58± 0.26a
0.63±0.21a
2.78± 0.23b
1.60±0.09a
1.94±0.31b
2.59±0.20a
0.81± 0.20a
0.57± 0.04a
1.13± 0.42a
2.82± 0.19a
3.29±0.12b
2.49
±0.07a
2.33±0.21b
0.52±0.20a
Results are mean ± standard deviation of mean of five samples.
All means in the same column with similar super scripts are not different from each other.
The significant difference was at p<0.05.
Table 3: Effect of treatment atherogenic and coronary risk index
Group/ Treatment
A (Normal control)
B (Calcium)
C
(Artemisinin
combination)
D (Artemisinin + Calcium)
Note:



Triglyceride (mmol/L)
1.69±0.07a
1.58±0.06a
2.11±0.21a
1.71±0.05a
Results are mean ± standard deviation of mean of five samples.
All means in the same column with similar super scripts are not different from each other.
The significant difference was at p<0.05.
Table 2: Effect of Treatment on Plasma lipid
Group/Treatment
Note:



Total cholesterol (mmol/L)
4.58±0.10a
2.61±0.12b
4.51±0.18a
5.12±0.56a
Atherogenic index
2.49±0.32a
3.54±0.26b
2.15±0.13a
Coronary risk index
0.65±0.22a
0.87±0.19a
0.60±0.02a
2.89±0.37a
0.259±0.01a
Results are mean ± standard deviation of mean of five samples.
All means in the same column with similar super scripts are not different from each other.
The significant difference was at p<0.05.
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